UVB breaks the B-ring of 7-dehydrocholesterol leading to the formation of pre-vitamin D3 that either undergoes thermal isomerization to D3 or photoisomerization to lumisterol (L3) in a 9?,10?-configuration. L3 is the major photoisomer formed after prolonged exposure of the skin to UVB energy, in a process that is irreversible. L3 has been considered to be metabolically and biologically inactive with its formation explaining why UVB induced cutaneous production of pre-D3 does not lead to systemic intoxication by D3. Recently we found that L3 is metabolized by CYP11A1 to 22(OH)L3, 24(OH)L3, 20,22(OH)2D3 and pregnalumisterol (pL) [collectively called (OH)nL3]. Uning LC/qTOF-MS we demonstrated the presence of L3 in human serum at levels higher than D3, and detectable levels of (OH)nL3 in the epidermis and serum. Furthermore, we found that (OH)nL3 inhibits the proliferation of human epidermal keratinocytes and that 20(OH)L3 stimulates the expression of genes associated with keratinocyte differentiation and protection against oxidative stress. Our preliminary molecular modeling predicts that the major (OH)nL3 products can act on ROR? and ROR? showing favorable docking scores in the ligand binding domain (LBD) of the receptors, similar to known natural ligands. Supporting these predictions we found that 20(OH)L3 inhibits ROR? and ROR? transactivation activities in a Tet-on CHO cell reporter system, as well as reducing (RORE)-LUC reporter activity in skin cells. Thus, we have discovered a new lumisterogenic biochemical pathway that is biologically relevant. To investigate its role in skin physiology and pathology we formulate the hypothesis that skin-derived L3 is enzymatically activated generating (OH)nL3 compounds which regulate epidermal barrier and photoprotective functions through interaction with ROR? and ROR?. This hypothesis will be tested via three aims. 1. Defining ROR? and ROR? as functional receptors for novel hydroxylumisterol derivatives ((OH)nL3); 2. Defining the phenotypic activities of (OH)nL3 in cultured epidermal keratinocytes and in human skin histocultured ex vivo; 3. Defining the relative roles of ROR? and ROR? in (OH)nL3-mediated regulation of the differentiation program and protective mechanisms against UVB radiation in the epidermis. Our highly mechanistic strategy combines techniques of primary epidermal and organ cultures, in silico methods, cell-based transcriptional studies coupled to the LBD of ROR?/?, the use of genetically modified cells, whole genome RNA sequencing with bioinformatics, and the techniques of biochemistry, chemistry and molecular biology. This proposal is a comprehensive, multi- disciplinary and state-of-the-art investigation. Its significance encompasses defining a role for a previously unrecognized pathway of activation of an important UV photoproduct, L3, in skin physiology and pathology. The practical outcome of the realization of aims 1-3 would be the use of the most potent (OH)nL3 as agent(s) that either enhance the epidermal barrier or prevent or reverse damage inflicted by noxious stressors and UVB. Establishing roles for RORs in these processes will define them as druggable pharmacological targets.